The present invention relates to electrophysiology and “patch clamping” for investigating ionic and molecular transport through cellular membranes via ion channels and, in particular, to a substrate providing a set of nano to microscale pores that may be readily sealed to cellular membranes. Ion channel investigation using patch clamps often plays an important role in drug discovery and preliminary drug screening or evaluation, for example, by providing a model that shows an effect of a drug on ion channels. Doing so can be useful for either avoiding adverse effects or for creating a positive therapeutic effect for the treatment of ion channel related diseases.
Drug screening can require a large number of ion channel measurements. Accordingly, in current practice, planar patch clamps are preferable because they allow parallelization of multiple samples on a substrate, often referred to as a wafer, chip, or well-plate, and facilitate measurement automation. Each sample has a cell or cell wall that is positioned so that an ion channel in the cell or cell wall is aligned with a pore at the sample site. The cell or cell wall is sealed to the patch clamp substrate in a manner that allows a small amount of electrical current to be used in performing ion channel investigations, typically by way of an extremely high resistance seal between the patch clamp substrate and the cell wall (a gigaohm seal or gigaseal). Gigaohm seals achieved using on-chip patch clamp procedures usually have electrical resistance values of about 1 gigaohm, with resistance values of up to about 5 gigaohms being achieved in some relatively rare instances.
Planar patch clamp substrates can be made from, for example, silicon, Teflon®, PDMS (polydimethylsiloxane), PSG (phosphosilicate glass), or glass. While such materials prove suitable for many planar patch clamp implementations, a single crystal quartz (quartz) material can be a particularly desirable for making planar patch clamp substrates. Quartz exhibits particularly high electrical insulating properties and is piezoelectric. Its unique electrical characteristics allow it to be used as a patch clamp substrate by providing very low levels of background noise while performing ion channel investigations. Furthermore, quartz exhibits particularly good mechanical characteristics such as, for example, good hardness, thermal stability, and chemical stability characteristics. Despite a general recognition of quartz's suitability for use as a patch clamp substrate, many of its desirable characteristics, such as hardness, make fabricating (micromachining) the pores in a quartz substrate rather difficult and/or time consuming.
Traditionally, micromachining of quartz is performed using a combination of lithography and reactive ion etching (RIE). However, RIE techniques require multiple steps and are relatively slow processes.
Another method of micromachining quartz is by way of direct laser beam ablation. During direct laser beam ablation, a high power density, short pulse width femtosecond laser beam is irradiated directly onto quartz. The nonlinear interaction between the ultrafast laser pulses and quartz, which has a band gap of about 9 eV, results in a cyclic multiphoton absorption and electron excitation between the ground and excited states. During this process, the initial excited electrons induce an avalanche ionization and generate a microplasma which ablates the quartz. However, since quartz has a wide band gap, this approach is also slow and is limited in terms of pore diameter and material thickness that can be achieved.
Recently, numerous advances have been made in micromachining of pores in non-quartz substrates, for example, by utilizing nanosecond lasers, such as excimer lasers instead of femtosecond lasers. Excimer lasers, which emit ultraviolet (UV) light, have been successfully implemented in relatively fast drilling procedures in non-quartz materials. However, quartz has excellent optical transmission over a large spectrum, from UV to infrared (IR), whereby it is transparent to light(s) in this spectrum. Since quartz is transparent to and therefore substantially unaffected by UV light(s), it has been widely accepted that excimer lasers are not usable for micromachining quartz.
Furthermore, although various patch clamping and other techniques have been developed and, at least to some extent, standardized for successfully modeling and investigating ion channel function voltage-sensitive (or voltage-gated) ion channels, in-depth investigation of yet other types of ion channels, such as mechanosensitive ion channels, remains at least somewhat frustrating and/or impracticable. Accordingly, numerous molecular mechanisms and their functionalities within mechanosensitive ion channels remain unknown, whereby cellular responses to mechanical stimuli remain some of the least understood of the known sensory mechanisms.